Site specific radiopharmaceuticals are ones whereby radionuclides attached to biologically active molecules are carried to predetermined locations in the body. Biomolecules which have been utilized to date include receptor specific ligands, metabolic tracers, and proteins such as antibodies and their fragments. As applied to the fields of radioimmunodetection and radioimmunotherapy, the major goals remain the early and sensitive detection of cancer, occult infection and thrombi, as well as more effective therapy.
The bifunctional chelate approach has proved to be of value for incorporation of metals into biologically active molecules such as proteins, see Benisek et al., J. Biol. Chem., Vol. 243, page 4267 (1968), Sundberg et al., Nature, Vol. 250, page 587 (1974), and Sundberg et al., J. Med. Chem., Vol. 17, page 1304 (1974). When an appropriate ligand is attached via a covalent bond to the protein, specific coordination of the metal to the ligand preferentially occurs, yielding a labeled product which generally exhibits enhanced in vivo stability, see Meares et al., Accts. Chem. Res., Vol. 17, page 202 (1984), Hnatowich et al., Int. J. Appl. Radiat. Isot., Vol. 33, page 327 (1982), and Paik et al., Int. J. Nucl. Med. Biol., Vol. 12, page 3 (1987). The success of this method for a particular purpose relies upon two principal factors. First, the bifunctional chelate must covalently bind to the protein under conditions which do not adversely affect the protein. In addition, it must possess a high affinity for the metal, so that the metal binds specifically to the chelate. In recent years, there has been much interest in the use of the bifunctional chelate approach for the radiolabeling of proteins with technetium-99m in the field of diagnostic Nuclear Medicine, see Fritzberg, A. R., Nucl. -Med., Vol. 26, page 7 (1987).
This technology has enabled detection of primary and metastatic tumors, using antibodies from monoclonal or polyclonal sources, both in animal models and in man. In the therapeutic arena, partial and total remission of several cancers including hepatomas, Hodgkins' disease and B cell lymphoma have been reported even for patients where other therapeutic regimens have failed. Although progress has been made in this multi-faceted research area, many significant problems remain. Optimum imaging protocols, appropriate dosimetry and background reduction techniques are just a few aspects which are the subject of continued research efforts. A major determinant for successful radioimmunodetection and radioimmunotherapy is the development of efficient, reproducible labeling methodologies yielding products in which the radionuclide is attached to the protein in a stable manner while maintaining protein viability.
For diagnostic imaging, technetium-99m (Tc-99m) possesses the best characteristics among currently available radionuclides. Its high photon yield per disintegration ensures good counting statistics with most protocols. The mono-energetic gamma photons are ideally suited for planar and single photon emission computed tomography (SPECT) instrumentation. These 140 KeV photons are soft enough to be adequately collimated while they are hard enough to penetrate overlying tissue for external detection with high sensitivity. The short half life (T.sub.1/2, 6 hours) and lack of particulate emissions generally result in low absorbed radiation dose. In addition, Tc-99m is inexpensive, approximately $1/mCi, and is widely available in generator form. Because of these properties, there is considerable interest in developing radiopharmaceuticals containing this radionuclide. Indeed, recent studies have demonstrated the applicability of Tc-99m to radioimmunodetection despite its short half life.
Of the radionuclides used for therapeutic applications, Re-186 (.beta.-.sub.max 1070 KeV; gamma 137 KeV, 9%; T.sub.1/2 90 hours) and Y-90 (.beta.-.sub.max 2290 KeV; gamma none; T.sub.1/2 64 hours), have been judged the best. These radionuclides possess strong .beta.- emissions capable of delivering high doses to tissues. An important advantage of Re-186 over other therapeutic radionuclides is the associated emission at practically the same energy as Tc-99m; thus, it is possible to follow its biodistribution with the same external scintigraphic equipment used for Tc-99m. Also, Re and Tc are congeners in the periodic table of elements and share certain similar chemical properties. This provides a rationale for designing therapeutic Re analogs of existing diagnostic Tc agents.
Early methods for labeling proteins with Tc-99m relied on the native protein to offer the stabilization needed for reduced Tc and are fraught with problems of weak non-specific labeling, colloidal contamination, protein denaturation and loss of label in vivo. Due to the advantages that the bifunctional chelate approach can provide, it is under investigation for the labeling of proteins with metallic radionuclides such as Tc-99m and Re-186. However, the bifunctional chelating agents (BCAs) used with other radionuclides have met with only limited success for Tc-99m and Re-186. For example, several attempts have been made to use diethylenetriaminepentaacetate (DTPA) coupled proteins to accept reduced Tc-99m. The major problem with proteins labeled in this manner is the lack of sufficient in vitro and in vivo stability. In our experience with Tc-99m labeled DTPA coupled to human immunoglobulin G (IgG), 40% of the radiolabel was lost from the protein within 3 hours in vitro. Furthermore, proteins compete with DTPA for reduced Tc which leads to non-specific binding of Tc to low affinity sites on the protein (Lanteigne and Hantowich, Int. J. Appl. Radiat. Isot., Vol. 35, pages 617-621 (1984)). In order to avoid the incorporation of Tc to weak binding sites, the labeling step can be performed in the presence of excess free DTPA ligand. However, this leads to low yields of Tc-bound protein and does not solve the problem of in vivo trans-chelation to other proteins. Similarly, an attempt to label DTPA coupled proteins with Re-186 also resulted in very low yields.
Some of the ligands which form complexes with technetium-99m are described in U.S. Pat. No. 4,638,051 and patents and publications cited therein.
It was desirable that a BCA be designed to meet a number of criteria. First, it must form a kinetically stable 1:1 complex with Tc-99m. Then, it must exhibit favorable exchange kinetics with a pre-formed labile Tc complex under mild labeling conditions. The BCA, as well as the reaction conditions used to couple it to the protein, or other sensitive biomolecules must be compatible with sensitive biomolecules such that direct conjugation of the ligand to protein via a covalent bond occurs under mild conditions.
The requirement for 1:1 stoichiometry between the ligand and Tc will ensure that no aggregation of the coupled protein would occur as a result of the complexation process. The strong chelating ligand should be able to extract reduced Tc-99m efficiently from the pre-formed Tc-99m complex as well as from any labile sites on protein. The rate of transchelation should be fast relative to the half-life of Tc-99m. Compatibility with the protein is desirable to ensure that the ligand itself does not cause denaturation of the protein. The strong BCA should directly couple to the protein without additional activation.
European Patent Application 0 188 256 discloses metal chelating compounds which are dithio-, diamino- or diamidocarboxylic acids or derivatives thereof and appears to have tried to claim a diaminodithiol (DADT) chelating ligand system. The compounds are intended for complexing with radionuclides.
U.S. Pat. No. 4,434,151 discloses the use of thiolactone as a coupling agent, but the agent is not internal in nature.